Investigations within this project concern the cell biology of rare human genetic disorders and normal and abnormal intracellular processes. The research goal is to gain insight into changes in molecular function that underlie various genetic metabolic disorders and work towards treatments for these illnesses. The research focuses on three groups of rare disorders: 1. Disorders of sialic acid metabolism. The key enzyme in the sialic acid biosynthesis pathway is UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Dominant mutations in the allosteric site of GNE cause sialuria, characterized by overproduction of sialic acid. Recessive mutations in GNE cause the neuromuscular disorder hereditary inclusion body myopathy (HIBM). Our group identified hypoglycosylation of alpha-dystroglycan as a possible underlying cause for the myopathy in HIBM, and found that polymorphisms in dystroglycan were not responsible for the variation in clinical phenotype. We also developed GNE-epimerase and GNE-kinase enzyme assays and demonstrated impaired GNE enzymatic activities in HIBM patients cells. We are currently characterizing a knock-in HIBM mouse model with to test several HIBM treatment options on these mice. We developed an allele specific real-time PCR method (patent application in progress; manuscript in preparation) to measure GNE allelic expression levels in both HIBM and sialuria. In addition, we are performing in vitro siRNA silencing experiments of the dominant, mutated allele in sialuria cells (manuscript in preparation). 2. Disorders of 3-methylglutaconic aciduria (3MGA) presenting with or without optic atrophy. We reported that optic atrophy is a common, under-recognized, clinical feature in many metabolic disorder. In 2001, our group isolated OPA3, a gene of unknown function, responsible for Costeff syndrome, which is characterized by 3MGA and optic atrophy. We are currently investigating OPA3 function, and we have identified a novel OPA3 isoform with a rare dual mitochondrial and peroxisomal localization. We also created zebrafish models for Costeff syndrome using antisense morpholino technology (manuscript in preparation). Investigations are ongoing into a candidate gene, OPA4, responsible for genetically unclassified patients with optic atrophy and 3MGA. 3. Disorders of intracellular vesicle sorting and formation. These disorders include Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syndrome, Griscelli syndrome, and other genetically unclassified disorders. Common clinical features are albinism due to defects in melanosomes and bleeding due to platelet defects. Our group assists in the investigations of known and unknown HPS-causing genes, with the goal of better understanding the biology of the disease. Our group assisted in cataloguing the clinical and genetic characteristics of the seven distinct subgroups of HPS. Collaborating research groups discovered several other HPS-related genetic mutations in mice, which prompted us to screen our unclassified group of human HPS patients for mutations in these genes. To study the effects of HPS mutations, we are performing confocal microscopy studies (using immuno-fluorescence, FRET, and live cell imaging) to examine defective intracellular trafficking and sorting of proteins and organelles in HPS cells. Such cells fail to transport certain lysosomal proteins to their correct destinations, and HPS gene products are involved in recognizing the specific vesicles that give rise to lysosome-like organelles.